Type IV pill (Tfp) are important determinants of bacterial virulence and biofilm formation which is the cause of many opportunistic and chronic bacterial infections. Tfp also mediate a form of bacterial surface motility known as social (S) gliding in Myxococcus xanthus and twitching in other bacterial species. The function of Tfp in surface motility correlates with their function in bacterial pathogenicity and biofilm formation. Mutations in genes essential for Tfp biogenesis and function lead to simultaneous defects in bacterial virulence, biofilm formation and Tfp-mediated motility. Our long term goal is to use M. xanthus S-motility as a model to study Tfp-mediated functions and signaling. S-motility itself also warrants further studies on its own merit because it is important to the developmental process of M. xanthus multicellular fruiting bodies. Besides Tfp, M. xanthus S-motility requires another cell surface component known as extracellular fibrils. Previous studies showed that the dif genes, encoding homologues of bacterial chemotaxis proteins, are central to the biogenesis or production of fibrils. Recent findings suggest that the pil genes which are required for Tfp biogenesis and function are also involved in the regulation of fibril biogenesis in M. xanthus. We propose to do the following. First, we will verify the requirement of fibril biogenesis for Tfp. We will further construct double mutants to examine the epistatic relationships in the regulation of fibril biogenesis among dif and pil genes as predicted by our hypothesis. Second, we will use yeast two-hybrid (Y2H) system and phosphorylation studies to examine if the Dif chemosensory-like proteins interact with one another physically and biochemically as their counterparts in bacterial chemotaxis and to identify any novel interactions that may exist among Dif. Third, it is unknown whether DifA, a homologue of methyl-accepting chemoreceptor proteins (MCP), localizes to specific subcellular regions and how DifA and the Dif pathway respond to stimulation. We will study DifA localization and the dependence of correct DifA localization on dif and pil genes by immunofluorescence microscopy. We will additionally construct and use NarX-DifA fusions to examine DifA modification and the signaling properties of the Dif pathway in response to stimulation by nitrate. Finally, Y2H and genetic screens will be carried out to identify new genes important for fibril regulation and biogenesis, especially downstream of Dif proteins.